Clinical laboratory characteristics and gene mutation spectrum of Ph‐negative MPN patients with atypical variants of JAK2, MPL, or CALR

Abstract Objective To evaluate the incidence, clinical laboratory characteristics, and gene mutation spectrum of Ph‐negative MPN patients with atypical variants of JAK2, MPL, or CALR. Methods We collected a total of 359 Ph‐negative MPN patients with classical mutations in driver genes JAK2, MPL, or CALR, and divided them into two groups based on whether they had additional atypical variants of driver genes JAK2, MPL, or CALR: 304 patients without atypical variants of driver genes and 55 patients with atypical variants of driver genes. We analyzed the relevant characteristics of these patients. Results This study included 359 patients with Ph‐negative MPNs with JAK2, MPL, or CALR classical mutations and found that 55 (15%) patients had atypical variants of JAK2, MPL, or CALR. Among them, 28 cases (51%) were male, and 27 (49%) were female, with a median age of 64 years (range, 21–83). The age of ET patients with atypical variants was higher than that of ET patients without atypical variants [70 (28–80) vs. 61 (19–82), p = 0.03]. The incidence of classical MPL mutations in ET patients with atypical variants was higher than in ET patients without atypical variants [13.3% (2/15) vs. 0% (0/95), p = 0.02]. The number of gene mutations in patients with atypical variants of driver genes PV, ET, and Overt‐PMF is more than in patients without atypical variants of PV, ET, and Overt‐PMF [PV: 3 (2–6) vs. 2 (1–7), p < 0.001; ET: 4 (2–8) vs. 2 (1–7), p < 0.05; Overt‐PMF: 5 (2–9) vs. 3 (1–8), p < 0.001]. The incidence of SH2B3 and ASXL1 mutations were higher in MPN patients with atypical variants than in those without atypical variants (SH2B3: 16% vs. 6%, p < 0.01; ASXL1: 24% vs. 13%, p < 0.05). Conclusion These data indicate that classical mutations of JAK2, MPL, and CALR may not be completely mutually exclusive with atypical variants of JAK2, MPL, and CALR. In this study, 30 different atypical variants of JAK2, MPL, and CALR were identified, JAK2 G127D being the most common (42%, 23/55). Interestingly, JAK2 G127D only co‐occurred with JAK2 V617F mutation. The incidence of atypical variants of JAK2 in Ph‐negative MPNs was much higher than that of the atypical variants of MPL and CALR. The significance of these atypical variants will be further studied in the future.


| INTRODUCTION
Classical Ph-negative myeloproliferative neoplasms (MPNs) are a type of clonal hematopoietic stem cell (HSC) disease.According to the latest WHO classification of hematopoietic and lymphoid tissues, mainly include three classical subtypes: polycythaemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF).5][6][7][8] The classical mutation of driver genes positive is one of the main diagnostic criteria for Ph-negative MPN.][11][12] Classical mutations of driver genes JAK2, MPL, and CALR in Ph-negative MPN are considered to be mutually exclusive. 13,142][23] At present, there is scant research on the Ph-negative MPN cohort where classical mutations and atypical variants of driver genes coexist.There has been no previous study on the gene mutation spectrum of Ph-negative MPN patients in which classical mutations and atypical variants of driver genes coexist.For the first time, we used high sensitivity next generation sequencing technology to study Chinese Ph-negative MPN patients, filling a gap in the field.
In this study, our target population was Ph-negative MPN patients with atypical variants of driver genes.We studied the co-existence of classical mutations and atypical variants in driver genes JAK2, MPL, and CALR, and explore the incidence, gene mutation spectrum, and clinical laboratory characteristics of Ph-negative MPN patients who are positive for classical mutations in driver genes and also have additional atypical variants of JAK2, MPL, or CALR.

| Study population
We collected a total of 359 cases diagnosed Ph-negative MPN with classical mutations of driver genes positive from August 2017 to October 2022.All were cases of initial diagnosis or first referral.The MPN diagnosis was in accordance with the 2017 version of the WHO classification of hematopoietic and lymphoid tumors. 24According to whether there were additional atypical variants of the driver genes, Ph-negative MPN patients were divided into two groups:

| Bone marrow pathological diagnosis analysis
Bone marrow trephine biopsy specimens are prepared for paraffin embedding, using H&E automatic stainer (Coverstainer, DAKO) for Hematoxylin and Eosin (HE) staining, manual method for reticular fiber and periodic acid schiff (BASO, China) staining.According to the 2017 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues, 24 OLYMPUS BX53 biological microscope is used for disease classification diagnosis and reticular fibrosis level assessment of Ph-negative MPN.

| Chromosomal karyotype analysis
Bone marrow specimens were treated with RPMI1640 culture medium (YUANDE, China), and after mitosis of nucleated cells, colchicine (0.4 μg/mL) was used to arrest cells in the metaphase.The cells were then treated with a hypotonic solution (0.4% KCl), and a fixing solution (methanol:glacial acetic acid = 3:1) was used to fix the cells.The cells were stained for R or G bands with Giemsa stain solution (10% Giemsa staining solution) after spreading on slides, and completed bands were scanned for chromosomes in metaphase using an automatic chromosome scanning device (M9120, BEION, China).At least 20 metaphases should be analyzed for chromosome inspection whenever possible.The results of karyotyping were described according to the 2020 International System for Human Cytogenetic Nomenclature (ISCN).

| Statistical analysis
All data were statistically analyzed using IBM SPSS 26.0 and R software (Version 4.2.2).For intergroup comparisons of quantitative data that conform to a normal distribution, single-factor analysis of variance or t-tests were used, and the results were expressed as means ± standard deviations.Non-parametric tests such as the Mann-Whitney U-test were used for non-normally distributed data, and the results were expressed as medians (ranges).For categorical data, chi-squared tests (n ≥ 40 and T ≥ 5) or Fisher's exact probability tests (n ≥ 40 and 1 ≤ T < 5; n < 40 or T < 1) were used for intergroup comparisons.Differences were considered statistically significant when p < 0.05.

| Patient cohorts and characteristics
Among the 359 Ph-negative MPN patients with positive classical mutations in driver genes, there were 304 patients without atypical variants of driver genes [PV, n = 115; ET, n = 95; and PMF, n = 94 (including 33 cases of Pre-PMF and 61 cases of Overt-PMF)], and 55 patients with atypical variants [PV, n = 19; ET, n = 15; and PMF, n = 21 (including 6 cases of Pre-PMF and 15 cases of Overt-PMF)].The clinical laboratory characteristics of these two types of Ph-negative MPN patients are shown in Table 1.Patients with atypical variants of driver genes accounted for 15% (55/359) of the total Phnegative MPN, including 14% (19/134) in PV, 14% (15/110) in ET, and 18% (21/115) in PMF.Among the 55 patients with atypical variants of driver genes, there were 28 males and 27 females, with a median age of 64 (range: 21-83) years old.The age of ET patients with atypical variants of driver genes was higher than ET patients without atypical variants [70 (28-80) vs. 61 (19-82), p = 0.03], the age of PV, Pre-PMF and Overt-PMF patients with atypical variants of driver genes was no significant difference than PV, Pre-PMF and Overt-PMF patients without atypical variants.The incidence of classical MPL mutations in ET patients with atypical variants of driver genes was higher than ET patients without atypical variants [13.3% (2/15) vs. 0% (0/95), p = 0.02], the incidence of other classical mutated driver genes was not significantly different between the Ph-negative MPN subgroup with T A B L E 1 Clinical and laboratory characteristics of 55 patients with atypical variants of driver genes MPN and 304 patients without atypical variants of driver genes MPN.   1).
All non-driver mutant genes in 359 Ph-negative MPN patients are categorized according to the function of the gene pathway, and the incidence of mutations is in descending order: chromatin modification genes, DNA methylation genes, signal transduction genes, transcriptional factor genes, RNA splicing genes, tumor suppressor genes and others (Figure S3).Analyze the occurrence rate of non-driver gene mutations that are simultaneously >5% in the group MPN with atypical variants of driver genes and the group MPN without atypical variants of driver genes, the incidence of SH2B3 and ASXL1 mutations was higher in the MPN with atypical variants of driver genes than in the MPN without atypical variants of driver genes (SH2B3: 16% vs. 6%, p < 0.01; ASXL1: 24% vs. 13%, p < 0.05), there were no significant differences between the other mutated genes (Table 2).

| DISCUSSION
In this study, we evaluated the incidence, gene mutation spectrum, and clinical laboratory characteristics in Phnegative MPN patients who were classical mutations positive in driver genes and carrying atypical variants of JAK2, MPL or CALR.We found that 15% of the Ph-negative MPN patients positive for classical mutations in driver genes carried atypical variants outside JAK2, MPL, or CALR, with the rate being 14% in PV, 14% in ET, and 18% in PMF.No patient carrying co-existing classical mutations in driver T A B L E 2 Compare the occurrence rate of non-driver gene mutations that are simultaneously >5% in the group MPN with atypical variants of driver genes and the group MPN without atypical variants of driver genes.

Mutation genes
Total  genes was found in this study, which is consistent with previous studies.However, we found that classical mutations of JAK2, MPL, or CALR can coexist with atypical variants of JAK2, MPL, or CALR in Ph-negative MPN patients.We discovered for the first time that the incidence of JAK2 atypical variants is higher than that of MPL atypical variants, and the incidence of MPL atypical variants is higher than that of CALR atypical variants among the atypical variants of driver genes.PMF had a greater variety of atypical variants of driver genes than PV and ET.
Schulze et al. 25 found that 15% (12/82) of JAK2 V617F positive patients also carry atypical variants in JAK2 or classical mutations/atypical variants in MPL, but did not discover classical mutations or atypical variants in JAK2 or MPL in patients positive for the classical mutation in CALR Exon9.In our study, 16% (45/288) of JAK2 V617F positive patients carry atypical variants in JAK2, MPL, or CALR.However, we found that 11% (6/56) of patients who were positive for classical mutations in CALR Exon9 also carried atypical variants in JAK2, MPL, or CALR.Also, we found that 25% (3/12) of patients positive for classical mutations in MPL Exon10 carried atypical variants in JAK2 or MPL.In PV, we not only found atypical variants in JAK2, but also in MPL and CALR.
In Western research on atypical variants of driver genes in Ph-negative MPN patients, the most common is JAK2 R1063H. 25,26However, in our study JAK2 R1063H was not found, which is speculated to be due to differences in population genetic background.The JAK2 R1063H is a germline variation and is an SNP (rs41316003).JAK2 R1063H shows a weak induction of the JAK-STAT signal in hereditary erythrocytosis, and when JAK2 R1063H and JAK2 E846D coexist, they jointly induce a stronger JAK-STAT signal activation.In this study, a total of 30 atypical variants of the driver genes were found, most of these variants have not been reported in previous studies on Ph-negative MPNs.The median VAF of these atypical variants in the 40% ~ 60% range may originate from a germline, including JAK2 G127D, JAK2 V392M, JAK2 K1030R, JAK2 D1118E, MPL c.1653 + 3G > A, MPL R525T, MPL X636W, CALR A17G, CALR I38M, CALR R177Q, CALR p.K391-E393deIKDE.Due to the highest incidence of JAK2 G127D, we detected the patient's hair follicles to confirm that JAK2 G127D is a germline variation.9] The COSMIC cancer database shows that this mutation site has been detected in central nervous system tumors (COSM9500826), the SIFT and Mutation Taster functional prediction databases suggest that this variant is harmful.The 1000G EAS population database shows that this variant has a proportion of 1.5% in the population, and the gnomAD exome EAS population database shows that this variant has a proportion of 1.95% in the population, but JAK2 G127D in our Ph-negative MPN cohort incidence (6.41%, 23/359) is significantly higher than the SNP.The frequent co-existence of JAK2 G127D with JAK2 V617F in our patient cohort warrants attention.
Gill et al. 29 found in a study of 101 cases of MPN-MF in Hong Kong, that 16 patients had JAK2 G127D mutations, the classical mutations co-existing with JAK2 G127D include JAK2 V617F , MPL W515L/K, CALR Exon9 del, which is somewhat different from our research results.In this study, all classical mutations of the driver gene that coexist with JAK2 G127D are JAK2 V617F .We found that MPN patients with the JAK2 G127D variation had a lower JAK2 V617F VAF than those without the JAK2 G127D variation.We suspect that JAK2 G127D and JAK2 V617F may have a "synergistic effect", and further verification is needed for how they play a role in the occurrence and progression of MPN disease.This is also our next research plan.
In Ph-negative MPN patients with atypical variants of driver genes, the top 10 mutation frequency were all signal transduction (classical and atypical JAK2, atypical MPL, SH2B3 and atypical CALR) and epigenetic related mutations (TET2, ASXL1, KMT2D, DNMT3A and EP300).We found that in Ph-negative MPN, mutations in SH2B3 and ASXL1 were significantly higher in patients with atypical variants of driver genes than in patients without atypical variants of driver genes, this finding requires further research.] Benton et al. 32 found that the co-existence of JAK2 V617F and JAK2 atypical variants was associated with an increased risk of leukemia transformation in MF patients.Therefore, we speculate that patients with additional atypical variants of driver genes may associate with a poorer prognosis.Because the patients were not dynamically followed up, so it was not possible to conduct a prognostic analysis.Patients with driver gene atypical variants may have different disease processes, and it is necessary to observe dynamics of the follow-up through patterns of large-scale patients or multi-center collaboration.Our next focus will be on the mechanism of SH2B3 and ASXL1 mutations in Ph-negative MPN with driver gene atypical variants.

| CONCLUSION
The results of this study suggest that the driver genes JAK2, MPL, and CALR may not be completely mutually exclusive for classical mutations and atypical variants.Ph-negative MPN patients with atypical variants of driver genes have a higher mutation rate of SH2B3 and ASXL1, which may indicate poor prognosis.Although most of the 30 atypical variants of driver genes found in this study are predicted to be harmful functional mutations by authoritative functional databases, the prediction results may not be consistent with the actual effect produced in the organism, thus further functional validation of these variants is needed, which is also our next work plan.Currently, although the discovery of additional atypical variants of driver genes has not affected the therapeutic strategies or disease diagnosis for MPN patients, whether the existence of double or multiple mutations of driver genes will cause differences in clinical manifestations or disease progression deserves clinical attention.How the atypical variants of driver genes play a role in the occurrence and development of MPN is still unclear, and their potential biological significance and impact on prognosis need further research.

( 1 )
There were 304 patients without atypical variants of driver genes [PV, n = 115; ET, n = 95; and PMF, n = 94 (including 33 cases of Pre-PMF and 61 cases of Overt-PMF)].(2) There were 55 patients with atypical variants of driver genes [PV, n = 19; ET, n = 15; and PMF, n = 21 (including 6 cases of Pre-PMF and 15 cases of Overt-PMF)].A focused analysis was conducted on the incidence, clinical laboratory characteristics and next generation sequencing results of 55 Ph-negative MPN patients, who were positive for classical mutations of JAK2, MPL, or CALR and also had atypical variants of JAK2, MPL, or CALR.

T A B L E 3
Location, variation VAF, and functional prediction of JAK2, MPL, SIFT: T represents benign variants, D represents deleterious variants; PolyPhen2: B represents benign variants; P represents possibly deleterious variants.

F I G U R E 2
The positions of the atypical amino acid variations on the exons of driver genes, (A) JAK2, (B) MPL, and (C) CALR.

F I G U R E 3
The co-existence relationship between mutant genes.(A) Chord diagram of classical mutations and atypical variants in the driver genes JAK2, MPL, and CALR.(B) The relationship between gene mutations in 55 Ph-negative MPN patients with atypical variants of the driver genes is shown, displaying all mutated genes and separating JAK2, MPL, and CALR into classical mutations and atypical variants.JAK2 classical mutations are further divided into JAK2 V617F and JAK2 Exon12, and JAK2 atypical variants are divided into JAK2 G127D and the other atypical JAK2 variants.Blue and red represent significant co-existence and mutual exclusion relationships, respectively, with the P value denoted by.*p < 0.05, **p < 0.01, ***p < 0.001.There is a co-existence relationship between JAK2 G127D and JAK2 V617F (p < 0.01).